1. Field of the Invention
The present invention is directed to a method for determining the chemical reactivity of a molecule. More specifically, it is directed at a method for determining the electrophilic reactivity of a molecule by a sequence of chemical additions and then quantitatively or qualitatively measuring the electrophilic properties. In particular the invention provides a high throughput toxicity screening method for pharmaceutically active molecules.
2. Technology Description
The current trend in the pharmaceutical industry is to shorten the time scale for all aspects of drug discovery. The impetus for this trend is to respond to the development of combinatorial chemistry techniques combined with a wide variety of in vitro biochemical and cell-based high-throughput screening (HTS) assays. The application of these early discovery methodologies has greatly increased the capacity demands of secondary discovery screening efforts including preclinical studies such as membrane permeability, metabolic stability, and drug-drug interactions.
For many different chemicals, including some drugs, the mechanism of cellular toxicity is via covalent interactions with cellular macromolecules. These reactions are usually dependent upon the facile reaction between an electrophilic carbon of the compound with a nucleophilic moiety of the macromolecule. Potential electrophiles may range in size and chemical structure and react with macromolecules through a variety of mechanisms. If the determination of cellular toxicity can be made as early as possible during the drug discovery process, this can have the effect of quickly reducing developmental costs on molecules which are potentially toxic.
Reduced glutathione (γ-glutamylcysteineglycine, GSH) is well known to interact with reactive compounds and thus plays a critical role in detoxification of electrophilic compounds in vivo. The reaction between GSH and electrophilic compounds may occur via non-enzymatic as well as enzymatic (glutathione S-transferase) processes. However for the uncatalyzed reaction between GSH and a series of electrophiles, the rates of reaction depend solely upon the chemical nature of each electrophile. In this light, the non-enzymatic conjugation between glutathione and an electrophile may be used as a surrogate marker to assess the potential reactivity of novel chemical entities (NCEs) with cellular macromolecules in vivo.
WO99/61910 discloses a high throughput, on-line, pulsed ultrafiltration-mass spectrometric method for determining whether a compound has predetermined characteristics that would make it suitable for a specific purpose, e.g., drug development. The method is used for rapid screening of drugs or other compounds to determine the extent of their metabolism and to characterize their primary metabolites. If reactive and potentially toxic metabolites are formed the metabolites can be reacted with glutathione and then detected on-line using mass spectrometry in a rapid assay to assess the potential for toxicity.
In the field of flow cytometry, determination of the presence of GSH by reaction with mercury orange has been suggested. See, for example, “A Flow Cytometric Assay for Intracellular Nonprotein Thiols Using Mercury Orange”, Cytometry 9, 529-532 (1988).
Despite the above teachings, there still exists a need in the art for a high-throughput compound independent assay and method to identify potential electrophilic compounds as early as possible in drug discovery.